Co-production of sugars and aroma compounds from tobacco waste using biomass-degrading enzymes produced by Aspergillus brunneoviolaceus Ab-10.

Biomass-degrading enzymes produced by microorganisms have a great potential in the processing of agricultural wastes. In order to produce suitable biomass-degrading enzymes for releasing sugars and aroma compounds from tobacco scraps, the feasibility of directly using the scraps as a carbon source for enzyme production was investigated in this study. By comparative studies of ten fungal strains isolated from tobacco leaves, Aspergillus brunneoviolaceus Ab-10 was found to produce an efficient enzyme mixture for the saccharification of tobacco scraps. Proteomic analysis identified a set of plant biomass-degrading enzymes in the enzyme mixture, including amylases, hemicellulases, cellulases and pectinases. At a substrate concentration of 100 g/L and enzyme dosage of 4 mg/g, glucose of 17.6 g/L was produced from tobacco scraps using the crude enzyme produced by A. brunneoviolaceus Ab-10. In addition, the contents of 23 volatile molecules, including the aroma compounds 4-ketoisophorone and benzyl alcohol, were significantly increased after the enzymatic treatment. The results provide a strategy for valorization of tobacco waste by integrating the production of biomass-degrading enzymes into the tobacco scrap processing system.

[Expression of a SARS-CoV-2 neutralizing nanobody in Trichoderma reesei].

Nanobodies derived from camelid single-chain antibodies have the advantages of being small, simple, highly soluble and stable. Nanobodies can be administered by inhalation and therefore is potentially valuable for the prevention and control of respiratory viruses. Trichoderma reesei is a food-grade protein expression host with a cellulase production capacity of up to 80 g/L, which can be employed for low-cost production of therapeutic proteins. In this study, a codon-optimized SARS-CoV-2 neutralizing nanobody Nb20 was expressed in T. reesei under a strong constitutive promoter Pcdna1. Nb20 protein was fused downstream of the N-terminal fragment of cellobiohydrolase Ⅰ, and the fusion protein can be intracellularly cleaved by the KEX2 protease to release Nb20. In a shake-flask fermentation using glucose medium, 47.4 mg/L Nb20 was detected in the culture after 48 h of cultivation. The expressed Nb20 showed the ability to interact with the receptor-binding domain of SARS-CoV-2 spike protein, suggesting that it can be used for the neutralization of SARS-CoV-2. The results indicate that T. reesei has the potential for recombinant production of nanobodies.

Efficient Constitutive Expression of Cellulolytic Enzymes in Penicillium oxalicum for Improved Efficiency of Lignocellulose Degradation.

Efficient cellulolytic enzyme production is important for the development of lignocellulose-degrading enzyme mixtures. However, purification of cellulases from their native hosts is time- and labor-consuming. In this study, a constitutive expression system was developed in Penicillium oxalicum for the secreted production of proteins. Using a constitutive polyubiquitin gene promoter and cultivating with glucose as the sole carbon source, nine cellulolytic enzymes of different origins with relatively high purity were produced within 48 h. When supplemented to a commercial cellulase preparation, cellobiohydrolase I from P. funiculosum and cellobiohydrolase II from Talaromyces verruculosus showed remarkable enhancing effects on the hydrolysis of steam-exploded corn stover. Additionally, a synergistic effect was observed for these two cellobiohydrolases during the hydrolysis. Taken together, the constitutive expression system provides a convenient tool for the production of cellulolytic enzymes, which is expected to be useful in the development of highly efficient lignocellulose-degrading enzyme mixtures.